ALPL-1 is a target for chimeric antigen receptor therapy in osteosarcoma

Osteosarcoma (OS) remains a dismal malignancy in children and young adults, with poor outcome for metastatic and recurrent disease. Immunotherapies in OS are not as promising as in some other cancer types due to intra-tumor heterogeneity and considerable off-target expression of the potentially targetable proteins. Here we show that chimeric antigen receptor (CAR) T cells could successfully target an isoform of alkaline phosphatase, ALPL-1, which is highly and specifically expressed in primary and metastatic OS. The target recognition element of the second-generation CAR construct is based on two antibodies, previously shown to react against OS. T cells transduced with these CAR constructs mediate efficient and effective cytotoxicity against ALPL-positive cells in in vitro settings and in state-of-the-art in vivo orthotopic models of primary and metastatic OS, without unexpected toxicities against hematopoietic stem cells or healthy tissues. In summary, CAR-T cells targeting ALPL-1 show efficiency and specificity in treating OS in preclinical models, paving the path for clinical translation.


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No statistical methods were used to determine the sample size. CAR T cells generated from different healthy donors were used in separated experiments. It was reasoned that CAR T cells from 3 to 6 healthy donors would give valid account for donor variability based on our past experience with inter-donor variability. For in vivo study, a power analysis was conducted to determine the minimum sample size required to test the study hypothesis (G*Power v3.1.9.7). For in vitro experiments, each experiment was performed at least twice, when possible, as independent experiment (N=2), and with two to six technical replicates (n=2-6). The sample size was sufficiently powered to detect a difference between the group by statistical test.
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Human embryonic kidney cell line HEK-293 was from our collection, originally from cell Biolabs Inc.. The OST-3 and OST-4 primary cell lines were generated from OS samples surgically resected at the Hospital Universitario Central de Asturias (Oviedo, Spain) after obtaining the approval of the Institutional Ethics Committee of the

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Sample preparation Extracellular staining: Single-cell suspensions were stained with fluorescent antibodies in PBS supplemented with 2% of fetal bovine serum (FBS) for 15 minutes at room temperature in the dark. Then, sample were washed in PBS with 2% FBS and resuspended in the same media. For indirect staining, the primary antibody labeling was followed by staining with a compatible fluorescent dye-conjugated secondary antibody.
Intracellular staining: For TNFα production, CAR T cells were co-cultured with tumor cells (Osteosarcoma and non osteosarcoma cells) in the presence of BD GolgiPlug and BD Golgistop for 6h. Subsequently, cells were harvested and stained extracellularly and intracellularly using the PerFix-nc kit according to the manufacturer's instructions (Beckman Coulter).
CTV proliferation assay: Initial FSC-H/SSC-H gates were drawn on the cell populations, excluding debris, followed by SSC-A/SSC-W gate to define single cells. A further gate SSC-H/GFP negative was drawn to exclude possible leftover debris from dead ALPL-1 positive target cells which were GFP positive. Boundary between positive and negative CTV cells was established using cells not labelled for CTV.

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